Novel bacterial strains, methods of preparing the same and use thereof in fermentation processes for L-lysine production

ABSTRACT

The invention provides novel microorganisms, methods for the production thereof and novel processes for the production of amino acids. Mutagenesis of parental bacterial strains and selection of an improved raffinate-resistant phenotype enables the isolation of strains with enhanced growth properties that produce larger amounts of amino acid. Microorganisms of the invention are produced from amino acid producing parental strains such as Corynebacterium or Brevibacterium, particularly preferred are parental strains that produce L-lysine.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to the fields of microbiology andmicrobial genetics. More specifically, the invention relates to novelbacterial strains, methods and processes useful for the fermentativeproduction of amino acids.

[0003] 2. Related Art

[0004] Following the recognition that Corynebacteria were useful for thefermentative production of amino acids (S. Kinoshita et al., Proceedingsof the International Symposium on Enzyme Chemistry 2:464-468 (1957)),the industrial production of L-lysine became an economically importantindustrial process. Commercial production of this essential amino acidis principally done utilizing the gram positive Corynebacteriumglutamicum, Brevibacterium flavum and Brevibacterium lactofermentum(Kleemann, A., et. al., “Amino Acids,” in ULLMANN'S ENCYCLOPEDIA OFINDUSTRIAL CHEMISTRY, vol. A2, pp.57-97, Weinham:VCH-Verlagsgesellschaft (1985)). These organisms presently account forthe approximately 250,000 tons of L-lysine produced annually.

[0005] The efficiency of commercial production of L-lysine may beincreased by the isolation of mutant bacterial strains which producelarger amounts of L-lysine. Microorganisms employed in microbial processfor amino acid production are divided into 4 classes: wild-type strain,auxotrophic mutant. regulatory mutant and auxotrophic regulatory mutant(K. Nakayama et al., in Nutritional Improvement of Food and FeedProteins, M. Friedman, ed., (1978), pp.649-661). Mutants ofCorynebacterium and related organisms enable inexpensive production ofamino acids from cheap carbon sources, e.g., mollasses, acetic acid andethanol, by direct fermentation. In addition, the stereospecificity ofthe amino acids produced by fermentation (the L isomer) makes theprocess advantageous compared with synthetic processes.

[0006] Given the economic importance of L-lysine production by thefermentive process, the biochemical pathway for lysine synthesis hasbeen intensively investigated, ostensibly for the purpose of increasingthe total amount of L-lysine produced and decreasing production costs(recently reviewed by Sahm et al., Ann. N. Y. Acad. Sci. 782:25-39(1996)). Entry into the lysine pathway begins with L-aspartate (see FIG.1), which itself is produced by transamination of oxaloacetate. Aspecial feature of C. glutamicum is its ability to convert the lysineintermediate piperidine 2,6-dicarboxylate to diaminopimelate by twodifferent routes, i.e., by reactions involving succinylatedintermediates or by the single reaction of diaminopimelatedehydrogenase. Overall, carbon flux into the pathway is regulated at twopoints: first, through feedback inhibition of aspartate kinase by thelevels of both L-threonine and L-lysine; and second through the controlof the level of dihydrodipicolinate synthase. Increased production ofL-lysine may be therefore obtained in Corynebacteria by deregulating andincreasing the activity of these two enzymes.

[0007] In addition to the biochemical pathway leading to L-lysinesynthesis, recent evidence indicates that the transportation of L-lysineout of cells into the media is another factor to be considered in thedevelopment of lysine over-producing strains of C. glutamicum. Studiesby Kramer and colleagues indicate that passive transport of lysine outof the cell, as the result of a leaky membrane, is not the soleexplanation for lysine efflux; their data suggest a specific carrierwith the following properties: (1) the transporter possesses a ratherhigh Km value for lysine (20 mM); (2) the transporter is an OH⁻ symportsystem (uptake systems are H⁺ antiport systems); and (3) the transporteris positively charged, and membrane potential stimulates secretion (S.Bröer and R. Krämer, Eur. J. Biochem. 202: 137-143 (1991).

[0008] Several fermentation processes utilizing various strains isolatedfor auxotrophic or resistance properties are known in the art for theproduction of L-lysine: U.S. Pat. No. 2,979,439 discloses mutantsrequiring homoserine (or methionine and threonine); U.S. Pat. No.3,700,557 discloses mutants having a nutritional requirement forthreonine, methionine, arginine, histidine, leucine, isoleucine,phenylalanine, cystine, or cysteine; U.S. Pat. No. 3,707,441 discloses amutant having a resistance to a lysine analog; U.S. Pat. No. 3,687,810discloses a mutant having both an ability to produce L-lysine and aresistance to bacitracin, penicillin G orpolymyxin; U.S. Pat. No.3,708,395 discloses mutants having a nutritional requirement forhomoserine, threonine, threonine and methionine, leucine, isoleucine ormixtures thereof and a resistance to lysine, threonine, isoleucine oranalogs thereof; U.S. Pat. No. 3,825,472 discloses a mutant having aresistance to a lysine analog; U.S. Pat. No. 4,169,763 discloses mutantstrains of Corynebacterium that produce L-lysine and are resistant to atleast one of aspartic analogs and sulfa drugs; U.S. Pat. No. 5,846,790discloses a mutant strain able to produce L-glutamic acid and L-lysinein the absence of any biotin action-surpressing agent; and U.S. Pat. No.5,650,304 discloses a strain belonging to the genus Corynebacterium orBrevibacterium for the production of L-lysine that is resistant to4-N-(D-alanyl)-2,4-diamino-2,4-dideoxy-L-arabinose2,4-dideoxy-L-arabinose or a derivative thereof.

[0009] More recent developments in the area of L-lysine fermentiveproduction in Corynebacteria involve the use of molecular biologytechniques to augment lysine production. The following examples areprovided as being exemplary of the art: U.S. Pat. Nos. 4,560,654 and5,236,831 disclose an L-lysine producing mutant strain obtained bytransforming a host Corynebacterium or Brevibacterium microorganismwhich is sensitive to S-(2-aminoethyl)-cysteine with a recombinant DNAmolecule wherein a DNA fragment conferring resistance toS-(2-aminoethyl)-cysteine and lysine producing ability is inserted intoa vector DNA; U.S. Pat. No. 5,766,925 discloses a mutant strain producedby integrating a gene coding for aspartokinase, originating fromCoryneform bacteria, with desensitized feedback inhibition by L-lysineand L-threonine, into chromosomal DNA of a Coryneform bacteriumharboring leaky type homoserine dehydrogenase or a Coryneform bacteriumdeficient in homoserine dehydrogenase gene.

[0010] Many process designed utilizing bacterial mutant strains aredesigned to weaken bacterial growth and hence to enhance the yield ofamino acid production through supplementation with other nutrients.Usually, mutants designed to improve the percent yield of an amino acidfrom substrates such as glucose will also lose their ability forvigorous growth like their wild type strains. Besides resulting in anoverall decrease in amino acid yield, these mutants also require morenutrients to support their growth, which can increase the cost in theproduction significantly.

[0011] Thus, there is a continuing need in the art for the developmentof novel amino acid producing bacterial strains that enable maximizedyields of a particular amino acid at a low cost of production. In viewof these problems, an alternative method comprises special mutants andmedia that is employed to increase the productivity and to decrease theingredient cost.

SUMMARY OF THE INVENTION

[0012] The invention provides generally for novel microorganisms withimproved raffinate resistance and improved growth properties, whichenables higher yields of amino acid to be produced.

[0013] A first object of the invention provides novel methods for theproduction of microorganisms with increased ability to produce aminoacids. In a first embodiment of the invention, a method is provided forthe production of a novel strain by way of mutagenesis of an aminoacid-producing, parental bacterial strain and subsequent selection forthe improved raffinate resistant strains of the invention. In a morespecific embodiment of the invention, the methods are drawn to aminoacid-producing, parental bacterial strains such as Corynebacterium andBrevibacterium. A particularly favored embodiment is drawn to a methodfor the production of an improved raffinate-resistant, amino acidproducing bacterial strain that is Brevibacterium which producesL-lysine.

[0014] Another object of the invention is drawn to novel bacterialstrains with improved raffinate-resistance, improved growthcharacteristics and that produce larger amounts of amino acid. In afirst embodiment, bacterial strains of the invention are produced by aprocess wherein a parental bacterial strain is subjected to mutagenesisand mutant progeny bacteria are selected for improvedraffinate-resistance, improved growth characteristics and improved aminoacid production. A more specific embodiment is drawn to novelCorynebacterium or Brevibacterium microorganisms with improvedraffinate-resistance, improved growth characteristics and improved aminoacid production. Particularly favored embodiments of the invention aredrawn to Brevibacterium that produce large amounts of L-lysine. Mostfavored embodiments are drawn to the strains ADM L63.148 (NRRL B-30059),ADM L64.132 (NRRL B-30060), ADM L69.53 (NRRL B-30061), ADM L69.74 (NRRLB-30062), and ADM L69.100 (NRRL B-30063), or mutants thereof.

[0015] A third object of the invention provides processes for theproduction of an amino acid comprising the steps of (a) culturing abacterium in a raffinate containing medium and (b) recovering the aminoacid from the culture media. In a preferred embodiment, the culturedbacteria of step (a) is obtained by a method in which an aminoacid-producing, parental bacterial strain is subjected to mutagenesisand progeny are selected for improved raffinate-resistance, improvedgrowth characteristics and improved production of an amino acid. Favoredembodiments are drawn to processes for the production of an amino acidthat utilize Corynebacterium or Brevibacterium. Particularly favoredembodiments of the invention for processes for the production of anamino acid utilize Brevibacterium that produce L-lysine.

[0016] It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are intended to provide further explanation of theinvention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1. A) A schematic presentation of the biochemical pathwayleading to L-lysine production in Corynebacterium; B) A schematicpresentation of the biochemical pathway leading to L-isoleucineproduction in Corynebacterium.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] 1. Definitions

[0019] In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided.

[0020] High Yield Derivative: As used herein, the term refers to strainof microorganism that produces a higher yield from dextrose of aspecific amino acid when compared with the parental strain from which itis derived.

[0021] Mutation: As used herein, the term refers to a single base pairchange, insertion or deletion in the nucleotide sequence of interest.

[0022] Operon: As used herein, the term refers to a unit of bacterialgene expression and regulation, including the structural genes andregulatory elements in DNA.

[0023] Parental Strain: As used herein, the term refers to a strain ofmicroorganism subjected to some form of mutagenesis to yield themicroorganism of the invention.

[0024] Phenotype: As used herein, the term refers to observable physicalcharacteristics dependent upon the genetic constitution of amicroorganism.

[0025] Raffinate: As used herein, the term refers to a wastestreamproduct from an ion-exchange operation for lysine recovery. Raffinatecontains a large amount of ammonia sulfate, L-lysine, other amino acids,salts, and carbohydrates such as isomaltose. Sterilization of araffinate-containing medium using heat treatment produces amino acidderivatives and other metabolic antagonists which cause the inhibitionof culture growth.

[0026] Heat sterilized raffinate-containing medium may be used to selectmicroorganisms, e.g., Brevibacterium or Corynebacterium, that areresistant to amino acid derivatives contained therein that inhibitculture growth; that are resistant to metabolic inhibitors containedtherein that inhibit culture growth and/or that are resistant todegradation products of lysine and/or precursors to lysine containedtherein that inhibit culture growth.

[0027] Relative Growth: As used herein, the term refers to a measurementproviding an assessment of growth by directly comparing growth of aparental strain with that of a progeny strain over a defined time periodand with a defined medium.

[0028] Mutagenesis: As used herein, the term refers to a process wherebya mutation is generated in DNA. With “random” mutatgenesis, the exactsite of mutation is not predictable, occurring anywhere in thechromosome of the microorganism, and the mutation is brought about as aresult of physical damage caused by agents such as radiation or chemicaltreatment.

[0029] 2. Mutagenesis of Parental Bacterial Strains

[0030] The invention provides methods for the production ofmicroorganisms that produce large amounts of an amino acid and haveimproved resistance to raffinate. Through the course of studies, it hasnow been found that ammonia sulfate which is required for the growth andamino acid biosynthesis may be replaced with raffinate, a wastestreamproduct from an ion-exchange operation of lysine recovery. Raffinatecontains a lot of ammonia sulfate, L-lysine, other amino acids, salts,and carbohydrates such as isomaltose. During heat treatment to sterilizethe medium, however, this raffinate medium produces a lot of amino acidderivatives and other metabolic antagonists which causes the inhibitionof growth for culture. To overcome this problem, a method was designedto select strains which can resist high levels of raffinate in themedium and increase their amino acid production.

[0031] Bacterial strains of the invention are preferably made by meansof mutagenesis of a parental bacterial strain followed by selection ofthe improved raffinate-resistant phenotype. Parental microorganisms maybe selected from any organism known in the art to be useful for thefermentative production of amino acids; favored parental microorganismsare Corynebacterium and Brevibacterium that produce an amino acid, andmost particularly favored organisms are Corynebacterium andBrevibacterium that produce L-lysine.

[0032] In a first embodiment, the invention provides a methods for theproduction of improved raffinate-resistant, amino acid-producing,bacterial strains comprising:

[0033] (a) subjecting a parental bacterial strain A to mutagenesis;

[0034] (b) contacting said mutagenized parental strain A with a mediumcontaining at least about 1% raffinate based on ammonia sulfate content;

[0035] (c) selecting raffinate-resistant bacterial strain B; and

[0036] (d) determining L-lysine production of said raffinate-resistantbacterial strain B.

[0037] The parental strain may be mutagenized using any randommutagenesis technique known in the art, including, but not limited to,radiation and chemical procedures. Particularly preferred is randomchemical mutagenesis, and most preferable is mutagenesis using asuitable agent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG).

[0038] General methods for mutagenesis and selection of novel bacterialstrains are well known in the art and are described, for example, in J.H. Miller, Experiments in Molecular Genetics, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1972); J. H. Miller, A ShortCourse in Bacterial Genetics, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1992); M. Singer and P. Berg, Genes & Genomes,University Science Books, Mill Valley, Calif. (1991); J. Sambrook, E. F.Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); P.B. Kaufman et al., Handbook of Molecular and Cellular Methods in Biologyand Medicine, CRC Press, Boca Raton, Fla. (1995); Methods in PlantMolecular Biology and Biotechnology, B. R. Glick and J. E. Thompson,eds., CRC Press, Boca Raton, Fla. (1993); and P. F. Smith-Keary,Molecular Genetics of Escherichia coli, The Guilford Press, New York,N.Y. (1989).

[0039] Strains of the invention have an improved raffinate resistantphenotype, which is determined by the concentration of raffinate, asmeasured by ammonium sulfate content, in the selection medium employed.In a first embodiment, phenotype selection may be done in a mediumcontaining at least about 1% raffinate. In a most preferred embodiment,microorganisms of the invention are selected in medium containing about5% raffinate. Other examples include at least about 2%, 3%, 4%, 5%, 6%,7%, and 8% raffinate containing medium for use in the selection ofimproved raffinate resistant strains.

[0040] The invention provides generally for novel microorganisms withimproved raffinate resistance and improved growth properties, whichenables higher yields of amino acid to be produced. An important elementor property of the methods, processes or microorganisms of the inventionis related to raffinate resistance.

[0041] Skilled artisans in the art of fermentative amino acid productionare familiar with the term “raffinate” as used herein. However, for thepurposes of more fully providing a detailed description of Applicants'invention, a definition of raffinate and a method for its production areprovided.

[0042] The term “raffinate” is most closely associated with the chemicalengineering field in the area of liquid-liquid extraction. The term isdefined in solvent refining as “that portion of the treated liquidmixture that remains undissolved and is not removed by the selectivesolvent” (Dictionary of Scientific and Technical Terms, Sybil P. Parker,ed., McGraw-Hill (1989)). As used herein, the term is associated withthe application of ion-exchange chromatography in the isolation of aminoacids. In an analogous fashion to the process of liquid-liquidextraction, the term raffinate as used in connection with ion-exhangechromatography refers to that portion of the liquid mixture that is notselectively bound by the chromatographic resin. More specifically, inconnection with the fermentative production of amino acids, theraffinate is that portion of the cell culture media that does not bindto the chromatographic column; raffinate is the broth effluent wastestream product generated during the ion-exchange chromatographicpurification of an amino acid. Typically, as used herein, raffinaterefers to the first waste stream product generated after the initialapplication of the growth media to the ion-exchange resin.

[0043] A variety of ion-exchange chromatographic methods may be utilizedfor the purification of amino acids. Typically, cation exchange resinsare utilized for the purification of lysine. Ion-exchange chromatographymay be done utilizing a fixed bed or simulated moving bed resin. Forexample, Van Walsem and Thompson describe a simulated moving bedtechnique for the isolation of lysine (Van Walsem, H. J. and Thompson,M. C., J. Biotechnology 59:127-132 (1997); U.S. Pat. Nos. 4,714,767 and5,684,190 describe the use of a fixed bed chromatographic technique forthe purification of amino acids and Wolfgang and Prior utilize anannular chromatograph to achieve a continuous mode of operation in theseparation of carbohydrates (Wolfgang, J. and Prior, A., SeparationScience and Technology 32:71-82 (1997)). Thus, the specificchromatographic method of generating raffinate may vary, but theunderlying principle defining raffinate remains constant.

[0044] For exemplary purposes only, Applicants provide in Example 5details for the production of raffinate for use as a cell growth mediumsupplement. As one skilled in the art would know, raffinate may bequalitatively characterized according to the specific amino acidproduced in the fermentation medium from which the raffinate isisolated; for example, raffinate may be known as lysine-raffinate whenisolated from lysine fermentation medium, glycine-raffinate whenisolated from glycine fermentation medium, isoleucine-raffimate whenisolated from isoleucine fermentation medium, etc. It will be readilyapparent to those skilled in the art that when the general termraffinate is used herein, the specific type of raffinate selected willdepend upon practitioner design.

[0045] The example provided herein is exemplary for the production ofraffinate, in particular for lysine-raffinate. As will be obvious tothose skilled in the art, other methods may be utilized in thegeneration of raffinate.

[0046] 3. Improved Raffinate Resistant Strains of the Invention

[0047] Another object of the invention is drawn to microorganisms thathave improved raffinate resistance and that produce an amino acid. Asone skilled in the art will know, such microorganisms may selected tohave improved resistance to any specific type of raffinate, for example,glycine-raffinate, valine-raffinate, isoleucine-raffinate,lysine-raffinate, etc. In a particularly preferred embodiment, themicroorganisms have improved resistance to lysine-raffinate.

[0048] In a specific embodiment of the invention, theraffinate-resistant microorganisms are produced by a process wherein:

[0049] (a) a parental bacterial strain A is subjected to mutagenesis;

[0050] (b) the mutagenized parental strain A is contacted with a mediumcontaining at least about 1% raffinate based on ammonia sulfate content;

[0051] (c) a raffinate-resistant bacterial strain B is selected; and

[0052] (d) amino acid production of said raffinate-resistant bacterialstrain B is determined.

[0053] Selection of parental bacterial strains, mutagenesis and theselection of microorganisms of the invention with improved raffinateresistance may be done as heretofore described.

[0054] A more specific embodiment of the invention is drawn toCorynebacterium or Brevibacterium; especially favored areCorynebacterium or Brevibacterium that produce L-lysine.

[0055] The invention also provides a Corynebacterium strain producing atleast about 10 g L-lysine/liter/in 24 hours when grown in a mediumcontaining at least about 1% raffinate.

[0056] A particularly favored embodiment of the invention is drawn to anL-lysine producing Corynebacterium strain, wherein said strain isselected from the group consisting of NRRL B-30059, NRRL B-30060, NRRLB-30061, NRRL B-30062, NRRL B-30063 and mutants thereof.

[0057] 4. Amino Acid Production and Purification

[0058] Other embodiments of the invention are drawn to processes for theproduction of an amino acid in a raffinate-containing medium. Suchprocesses involve (a) the culturing of an improved raffinate resistantbacterial strain and (b) recovery of the amino acid from culture media.

[0059] In a first specific embodiment, the invention provides a processfor the production of an amino acid comprising:

[0060] (a) culturing a bacterial B strain in a medium containingraffinate, whereby said strain is obtained by the following method:

[0061] (i) selecting a parental bacterial strain A that produces anamino acid;

[0062] (ii) subjecting said parental strain A to mutagenesis;

[0063] (iii) selecting an improved raffinate-resistant bacterial strainB; and

[0064] (b) recovering the amino acid from the culture media.

[0065] Selection of parental bacterial strains, mutagenesis and theselection of microorganisms of the invention with improved raffinateresistance may be done as heretofore described.

[0066] In preferred embodiments of the invention, other processes aredrawn to parental strains selected from the group consisting of L-lysineproducing Corynebacterium and Brevibacterium microorganisms, and a mostpreferred embodiment of the invention is drawn to a parental strain thatis Brevibacterium that produces the amino acid L-lysine.

[0067] The processes of the invention may further vary by way of thespecific method of culturing the microorganisms of the invention. Thus,a variety of fermentation techniques are known in the art which may beemployed in processes of the invention drawn to the production of aminoacids.

[0068] Illustrative examples of suitable carbon sources include, but arenot limited to: carbohydrates, such as glucose, fructose, sucrose,starch hydrolysate, cellulose hydrolysate and molasses; organic acids,such as acetic acid, propionic acid, formic acid, malic acid, citricacid, and fumaric acid; and alcohols, such as glycerol.

[0069] Illustrative examples of suitable nitrogen sources include, butare not limited to: ammonia, including ammonia gas and aqueous ammonia;ammonium salts of inorganic or organic acids, such as ammonium chloride,ammonium phosphate, ammonium sulfate and ammonium acetate; and othernitrogen-containing, including meat extract, peptone, corn steep liquor,casein hydrolysate, soybean cake hydrolysate and yeast extract.

[0070] Generally, amino acids may be commercially produced from theinvention in fermentation processes such as the batch type or of thefed-batch type. In batch type fermentations, all nutrients are added atthe beginning of the fermentation. In fed-batch or extended fed-batchtype fermentations one or a number of nutrients are continuouslysupplied to the culture, right from the beginning of the fermentation orafter the culture has reached a certain age, or when the nutrient(s)which are fed were exhausted from the culture fluid. A variant of theextended batch of fed-batch type fermentation is the repeated fed-batchor fill-and-draw fermentation, where part of the contents of thefermenter is removed at some time, for instance when the fermenter isfull, while feeding of a nutrient is continued. In this way afermentation can be extended for a longer time.

[0071] Another type of fermentation, the continuous fermentation orchemostat culture, uses continuous feeding of a complete medium, whileculture fluid is continuously or semi-continuously withdrawn in such away that the volume of the broth in the fermenter remains approximatelyconstant. A continuous fermentation can in principle be maintained foran infinite time.

[0072] In a batch fermentation an organism grows until one of theessential nutrients in the medium becomes exhausted, or untilfermentation conditions become unfavorable (e.g., the pH decreases to avalue inhibitory for microbial growth). In fed-batch fermentationsmeasures are normally taken to maintain favorable growth conditions,e.g., by using pH control, and exhaustion of one or more essentialnutrients is prevented by feeding these nutrient(s) to the culture. Themicroorganism will continue to grow, at a growth rate dictated by therate of nutrient feed. Generally a single nutrient, very often thecarbon source, will become limiting for growth. The same principleapplies for a continuous fermentation, usually one nutrient in themedium feed is limiting, all other nutrients are in excess. The limitingnutrient will be present in the culture fluid at a very lowconcentration, often unmeasurably low. Different types of nutrientlimitation can be employed. Carbon source limitation is most often used.Other examples are limitation by the nitrogen source, limitation byoxygen, limitation by a specific nutrient such as a vitamin or an aminoacid (in case the microorganism is auxotrophic for such a compound),limitation by sulphur and limitation by phosphorous.

[0073] Methods for the recovery and purification of amino acids,particularly L-lysine, are well known to those skilled in the art.Typically, an amino acid may be recovered from the growth medium bycation exchange, after centrifugation and filtration to remove cells.U.S. Pat. No. 5,684,190 describes the recovery of an amino acid such asL-lysine that involves (1) passage of the amino acid containing aqueoussolution over a primary cation exchange resin to absorb the amino acidonto the resin at a pH lower than its isoelectric point, subsequentlyfollowed by elution of the amino acid by increasing the pH with ammoniumhydroxide to produce a first solution; and (2) passage of the firstsolution over a secondary cation exchange resin in a similar fashion tofurther eliminate impurities.

[0074] Another example may be provided by U.S. Pat. No. 4,714,767, whichprovides a process for separating basic amino acids from an aqueoussolution using cation exchange resin towers in series. The processcomprises repetitive adsorption and elution steps in sequence, whereinthe washing water employed in the absorption and elution steps isobtained by recycling the latter portion of a liquid discharged from afirst tower absorption step or elution step in a subsequent cycle.

[0075] Eluants obtained from such cation exchange isolation proceduresmay be concentrated by evaporation, which additionally provides for theelimination of ammonia. The amino acid may then be crystallized fromsolution with hydrochloric acid, producing for exampleL-lysine.HCl.2H₂O. After centrifugation or filtration, the isolatedL-lysine crystals are dried.

EXAMPLES Example 1 Mutagenesis, Screening And Selection for ImprovedRaffinate Resistant Microorganisnis

[0076] The lysine producing strains such a T125, L58.23, and 96T116,whose growth is inhibited by higher concentrations of raffinate, weresubjected to mutagenesis, and mutants showing resistance to higherconcentrations of raffinate were recovered. For mutagenesis, bacterialcultures were grown to mid-log phase in medium B (Table 1), pelleted bycentrifugation and resuspended in 2 mL of filter-steriled TM buffer in a15 ml polypropylene conical tube (Tris.HCL 6.0 g/L, maleic acid 5.8 g/L,(NH₄)2SO₄1.0 g/L, Ca(NO₃)₂ 5 mg/L, MgSO₄.7H₂O 0.1 g/L, FeSO₄.7H₂O 0.25mg/L, adjusted to pH 6.0 using KOH). The 2 mL cell suspension was mixedwith 50 μL of a 5.0 mg/L solution of N′-nitro-N-nitrosoguanidine (NTG),then incubated at 30° C. for 30 minutes. An untreated cell suspensionwas similarly incubated as a control for estimating the kill rate. Afterincubation, 10 mL of TM buffer was added to each tube, and the cellswere pelleted by centrifugation, washed twice in TM buffer, andresuspended in 4.0 mL of 0.1 M NaH₂PO₄ (phosphate buffer) adjusted to pH7.2 using KOH. The washed cell suspensions were further diluted inphosphate buffer, and aliquots were spread on plates of medium A (Table1). After incubation at 30° C. for 4-6 days, colonies growing on mediumA agar were picked and tested for improved potential to produce L-lysinefrom dextrose in shaker flasks and fermentors.

Example 2 The Growth of Strains in Raffinate Media

[0077] For each tested strain (Table 2), 0.1 mL of frozen culture wasinoculated into a 250 mL baffled flask containing 20 mL raffinate mediumC (Table 1), then incubated for 18 hours at 30° C., at 240 rpm. Afterincubation, 50 μl of culture was removed and diluted to a ratio of 1:100in 0.1 N HCl solution. The optical density (OD) of the diluted samplewas measured at 660 nm with a spectrophotometer. The results are shownin Table 2. All strains with improved raffinate resistance (RF),L63.148, L64.132, L69.53, and L69.74, grew better (higher OD) than theirparental strains, 108T125, LS8.23, and 96T116, in the raffinate mediumC.

Example 3 Dextrose Consumption, Growth, and Lysine Production in ShakerFlask Fermentation

[0078] For each strain, 0.1 mL of a frozen culture was inoculated into a250 mL baffled flask containing 20 mL of seed medium C and incubated for18 hours at 30° C., 240 rpm. Two mL of seed culture were used toinoculated 20 mL of fermentation medium D in a 250 mL baffled flask. Theflasks were then shaken for 24 hours at 30° C. and 240 rpm. After 24hours of fermentation, samples were removed for analysis. To measuredextrose concentrations, 100 μL of sample were removed and diluted 1:50with deionized (DI) water and measured with a YSI biochemistry analyzer(Yellow Springs Instrument Co. Inc.). L-lysine concentrations weredetermined by HPLC. Optical density measurements were taken to measuregrowth as described in Example 2. Results are presented in Table 3; allraffinate resistant strains, L63.148, L64.132, L69.53 and L69.74, grewbetter, used dextrose more efficiently, and produced more L-lysine thantheir parent strains, 108T125, L58.23, and 96T116.

Example 4 Growth and L-Lysine Production in Bench Scale Fermentors

[0079] Bench scale fermentations were set up using a two stage inoculumprotocol. The first stage media was composed of 50.0 g/l sucrose, 3.0g/l K₂HPO₄, 3.0 g/l urea, 0.5 g/l MgSO₄-7H₂O, 30.0 g/l soy peptone, 5.0g/l yeast extract, 0.765 mg/l biotin, 3.0 mg/l thiamine HCl, and 0.125g/l niacinamide. A 2 liter baffled shake flask containing 500 mls ofthis media was inoculated with the culture and incubated at 30° C. and250 rpm for 19 hrs. At this point, 22.5 mls of the mature first culturewas used to inoculate the second stage inoculum media.

[0080] The second stage inoculum was prepared with 3000 mls of medium ina 6.6 liter fermentor. The medium formulation was 20.0 g/l (db) cornsteep liquor, 10.0 g/l ammonium sulfate as raffinate, 12.0 mg/lMnSO₄—H₂O, 3.0 mg/l biotin, 3.0 g/l thiamine HCl, 125 mg/l niacinamide,0.5 mls l antifoam, and 60 g/l dextrose, sterilized separately as a 360g/l solution and added to the fermentorjust prior to inoculation. Thefermentor was operated at 32° C., 1.2 vvm air, 600 rpm, and a pH controlpoint of 7.2. pH control was accomplished by the addition of NH₃ orNH₄OH. After 18-20 hrs the inoculum was considered mature and used toinoculate the production stage vessel.

[0081] Production stage medium was composed of 40 g/l (db) corn steepliquor, 20 g/l ammonium sulfate as raffinate, 12.0 mg/l MnSO₄—H₂O, 0.75mls/l antifoam and 12 g/l dextrose, sterilized separately as a 250 g/lsolution and added just prior to inoculation. Media formulation wasbased on a 2.1 liter initial volume which includes 500 mls of maturesecond stage broth as inoculum. Operating parameters were the following:32° C., 2.1 vvm air, and an initial and control point pH of 7.2. pHcontrol was again done with NH₃ or NH₄OH. Agitation was initially 600rpm, increased to 700 rpm at 9 hrs culture time and 900 rpm at 19 hrsculture time. The fermentation was fed on demand, as indicated by pHincreases due to dextrose depletion, a mixture of dextrose and ammoniumsulfate. The feed was prepared by sterilizing separately 2310 g dextrose+800 mls water and a volume of raffinate containing a total of 176 g ofammonium sulfate, then combining the two solutions upon cooling toambient temperature. Total fermentation time was 48 hrs. The vessel sizewas the same as that used for the second stage inoculum development.

[0082] Results of an experiment comparing the parent strain to the abovedescribed isolates in bench scale fermentation are presented in Table 4.

Example 5 Production of Raffinate

[0083] As previously described, raffinate may be qualitativelycharacterized according to the specific amino acid produced in thefermentation medium from which the raffinate is isolated. The exampleprovided herein is for the production of lysine-raffinate. However, oneskilled in the art would know, other types of raffinate, e.g., valine-or isoleucine-raffinate, etc., may be similarly produced by simplystarting with the appropriate fermentation broth, e.g., valine orisoleucine fermentation broth, etc.

[0084] As a first step in the production of lysine-raffinate, lysinefermentation broth is diluted to a lysine concentration of 65.5 g/l.After ultrafiltration to generate a permeate with a lysine concentrationof 40.3 g/l, the permeate is then concentrated to 123 g/l lysine with atotal dry solids concentration of 207 g/l.

[0085] The permeate concentrate is then fed into a chromatographicseparation system, for example I-SEP or C-SEP produced by AdvancedSeparation Technologies Incorporated (St. Petersburg, Fla.). Ionexchange chromatographic separation systems are commonly known in theart, as exemplified by U.S. Pat. Nos. 4,808,317 and 4,764,276, which areincorporated herein by reference. The waste effluent obtained therefromis considered the “dilute lysine-raffinate” solution. The dilutelysine-raffinate solution has a pH of 5.1 and it contains 34.3 g/lammonium sulfate and 2.8 g/l lysine with a total solids level 67 g/l.

[0086] The dilute lysine-raffinate solution is concentrated to 295 g/ltotal solids. Quantitated components of this “concentratedlysine-raffinate” solution include the following: 137.9 g/l ammoniumsulfate, 14.8 g/l lysine, 8.7 g/l valine, 8.1 g/l alanine, 2.4 g/llactic acid and 2.2 g/l acetic acid. This concentrated lysine-raffinatesolution is used in media preparation.

TABLES

[0087] The following tables are referenced in the Examples section.TABLE 1 Media Employed in Examples 1, 2, and 3 Ingredients (amount/L) AB C D Glucose 20 g   30 g 68 g Sucrose 50 g L-Alanine 0.5 g 0.5 gL-Methionine 0.5 g 0.5 g L-Threonine 0.25 g 0.25 g Biotin 0.05 mg 0.756mg 0.003 g 0.405 mg Thiamine 0.2 mg 0.003 g 0.003 g Niacinamide 0.05 g0.125 g 0.125 g Polypeptone Peptone (BBL) 20 g Beef Extract (BBL) 5 gCorn Steep Liquor¹   20 g Raffinate² 60 g   10 g 40 g Urea 2.5 g 3 g 50g Amonia Sulfate 10 g K₂HPO₄ 3 g KH₂PO₄ 1 g MgSO₄ · 7H₂O 0.4 g 0.5 gMnSO₄ · H₂O 0.01 g  0.01 g 0.01 g NaCl 1 g FeSO₄ · 7H₂O 0.01 g CaCO₃  50 g 50 g Agar 15 g pH (before autoclave) 7.2 7.3 7.4 7.4

[0088] TABLE 2 The Growth of Strains in Medium C Containing RaffinateStrain 108T125 L63.148 L58.23 L64.132 96T116 L69.53 L69.74 Type Wild¹RF² Wild RF Wild RF RF OD₆₆₀ 15.9 27.4 27.1 34.5 22.2 31.6 30.3

[0089] TABLE 3 The Dextrose Consumption (Dex), Growth (OD₆₆₀), andLysine Production (Lys) of Strains in 24 hr Shaker Flask Fermentation inMedium D Strain 108T125 L63.148 L58.23 L64.132 96T116 L69.53 L69.74 TypeWild RF Wild RF Wild RF RF Dex, g/L 25.9 66.7 40.6 68.8 45.8 78.8 76.6OD₆₆₀ 20.5 43.2 26.3 47.9 30.5 47.4 42.8 Lys, g/L  9.4 18.8 14.1 23.315.5 24.6 23.2

[0090] TABLE 4 Parent and Progeny Comparison of Growth (OD660) andL-lysine Production in 6.6 1 fermentors Strain OD @ 660 nm TotalProduct¹ g lysine/1/hr² 96T116 Wild 83.8 583 g 5.78 L69.53 RF 112.5 776g 7.70 L69.74 RF 122.4 807 g 8.01 L69.100 RF 93.3 745 g 7.48

What is claimed is:
 1. A method for the production of an improvedraffinate-resistant, amino acid producing bacterial strain B comprising:(a) subjecting a parental bacterial strain A to mutagenesis; (b)contacting said mutagenized parental strain A with a medium containingat least about 1% raffinate based on ammonia sulfate content; (c)selecting a raffinate-resistant bacterial strain B; and (d) determiningamino acid production of said raffinate-resistant bacterial strain B. 2.The method of claim 1, wherein said parental bacterial strain issubjected to random chemical mutagenesis.
 3. The method of claim 1,wherein said parental bacterial strain is selected from a groupconsisting of: (a) Corynebacterium sp.; (b) Brevibacterium sp.; (c)Escherichia coli; and (d) Bacillus sp.
 4. The method of claim 1, whereinsaid bacterial strain B produces an amino acid selected from the groupconsisting of: (a) glycine; (b) alanine; (c) methionine; (d)phenylalanine; (e) tryptophan; (f) proline; (g) serine; (h) threonine;(i) cysteine; (j) tyrosine; (k) asparagine; (l) glutamine; (m) asparticacid; (n) glutamic acid; (o) lysine; (p) arginine; (q) histidine; (r)isoleucine; (s) leucine; and (t) valine.
 5. The method of claim 1,wherein said parental bacterial strain is Corynebacterium sp. producingL-Lysine.
 6. A bacterial strain B that produces an amino acid producedby a process wherein: (a) a parental bacterial strain A is subjected tomutagenesis; (b) the mutagenized parental strain is contacted with amedium containing at least about 1% raffinate based on ammonia sulfatecontent; (c) raffinate-resistant bacterial strain B is selected fromsaid mutagenized parental strain; and (d) amino acid production of saidbacterial strain B is determined.
 7. The bacterial strain of claim 6,wherein the parental bacterial strain A is selected from a groupconsisting of the following: (a) Corynebacterium sp.; (b) Brevibacteriumsp.; (c) Escherichia coli; and (d) Bacillus sp.
 8. The bacterial strainof claim 7, wherein said bacterial strain B produces an amino acidselected from the group consisting of: (a) glycine; (b) alanine; (c)methionine; (d) phenylalanine; (e) tryptophan; (f) proline; (g) serine;(h) threonine; (i) cysteine; (j) tyrosine; (k) asparagine; (l)glutamine; (m) aspartic acid; (n) glutamic acid; (o) lysine; (p)arginine; (q) histidine; (r) isoleucine; (s) leucine; and (t) valine. 9.A Corynebacterium sp strain producing at least about 10 g/l L-lysine in24 hours when grown in a medium containing at least about 1% raffinate.10. A Brevibacterium strain producing at least about 10 g/l L-lysine in24 hours when grown in a medium containing at least about 1% raffinate.11. An L-lysine producing bacterial strain, wherein said strain isselected from the group consisting of: (a) NRRL B-30059; (b) NRRLB-30060; (c) NRRL B-30061; (d) NRRL B30062; (e) NRRL B-30063; and (f)mutants of (a), (b), (c), (d) or (e).
 12. The strain of claim 11,wherein said strain is NRRL B-30059.
 13. The strain of claim 11, whereinsaid strain is NRRL B-30060.
 14. The strain of claim 11, wherein saidstrain is NRRL B-30061.
 15. The strain of claim 11, wherein said strainis NRRL B-30062.
 16. The strain of claim 11, wherein said strain is NRRLB-30063.
 17. A process for the production of an amino acid comprising:(a) culturing a bacterium B in a medium containing raffinate, wherebysaid strain is obtained by the following method: (i) selecting aparental bacterial strain A that produces an amino acid; (ii) subjectingsaid parental strain to mutagenesis; (iii) selecting from saidmutagenized parental strain, an improved raffinate-resistant bacterialstrain B; and (b) recovering the amino acid from the culture media. 18.The process of claim 17, wherein the media concentration of raffinate isat least about 1% based on ammonia sulfate content.
 19. The process ofclaim 17, wherein the amount of L-lysine produced is at least about 10g/l L-lysine in 24 hours.
 20. The process of claim 17, wherein themedium concentration of raffinate is at least about 1% based on ammoniasulfate content and the amount of L-lysine produced is at least about 10g/l L-lysine in 24 hours.
 21. The process of claim 17, wherein theraffinate concentration is about 5% based on ammonia sulfate content andthe amount of L-lysine produced is at least about 10 g/l L-lysine in 24hours.
 22. The process of claim 17, wherein bacterium B is selected fromthe group consisting of: (a) Corynebacterium sp.; (b) Brevibacteriumsp.; (c) Escherichia coli; and (d) Bacillus sp.
 23. The process of claim22, wherein the bacterium B is Corynebacterium sp. selected from thegroup consisting of: (a) NRRL B-30059; (b) NRRL B-30060; (c) NRRLB-30061; (d) NRRL B-30062; and (e) NRRL B-30063; and (f) mutants of (a),(b), (c), (d) or (e).